The purpose is , determining whether amino acids or proteins are present in solution and amino acids are carrying a free amine group by using biuret test, ninhydrin test, xanthoproteic test and heavy metal precipitation test.
Proteins are organic compounds made of amino acids.The amino acids joined together by the peptide bonds.Proteins have three dimensional structures. Primary structure is the amino acid sequence.Secondary structure , alpha helix, beta sheet . Secondary structures are local, many regions of different secondary structure can be present in the same protein molecule.
It regularly repeats local structures stabilized by hydrogen bonds. Tertiary structure , is generally stabilized by nonlocal interactions, most commonly the formation of a hydrophobic core, but also through salt bridges, hydrogen bonds, disulfide bonds, and even post-translational modifications.Quaternary structure , formed by several protein Molecules usually called protein subunits.(1)
Figure 1 : Structure of proteins
There are several quantitative tests for determining whether amino acids or proteins are present in solution.These tests are specific for the presence of peptide bonds,certain types of side chains and the type of secondary structure present.Biuret test determines if there is 2 or more peptide or not.The biuret reagent is made of sodium hydroxide and copper sulfate. The blue reagent turns violet in the presence of proteins, and changes to pink when combined with short-chain polypeptides. (2)
Figure 1 : Biuret test
Ninhydrin detects if there is ammonia or primary, secondary amines or not.In ninhydrin test , if reacts with these free amines, a deep blue or purple color will observed.
Xanthoproteic test, determines benzene and benzene derivatives such as tyrosine, tyrptophane which are contains activated benzene rings and readily undergo nitration and phenyl alanine which is also contains benzene ring but not activated and not readily undergo nitration.Also the product will be yellow colored.(3)
Heavy metal test , makes ions to denaturate proteins.There are similar reagent with biuret test and it uses AgNO3 and iron(III) .
3. APPARATUS :
3.1. Equipment :
Test tubes ( 15 mL falcon) * 16
Test tube rack
Beaker 250 mL
3.2. Chemicals :
2% glycine solution
2% gelatin solution
2 % albumin solution
2 % tyrosine solution
2.5 M NaOH solution
5 % copper sulfate
5 % silver nitrate
5 % iron (III) nitrate
4. PROCEDURE :
5 tubes were labeled as 1,2,3,4,5 according to the scheme below:
For biuret test ; 1 mL of each solution in 1,2,3 and 5 was placed except casein and casein was put and 1 mL water was added in test tube 4.
7-10 drops of 2.5 M NaOH was added and 2 drops of 5% copper sulfate solution was added on each of the samples.
Observations were recorded.
For ninhydrin test ; another 5 test tubes were labeled.
7-10 drops of ninhydrin reagent was added to each test tube and heated in a water bath for 5 minutes.
Observations were recorded.
For xanthoproteic test ; another 5 test tubes were labeled.
10-15 drops of nitric acid was added to each test tube and heated in a water bath for 5-10 minutes.
For heavy metal precipitation test ; 2 mL milk was placed in 3 separate tubes and labeled as 1,2,3 .
10 drops of 5 % silver nitrate was added to test tube 1, 10 drops of 5 % iron ( III) nitrate to test tube 2 and 10 drops of sodium nitrate to test tube 3.
Observatios were recorded.
5. OBSERVATIONS :
For biuret test :
Blue ( light)
Purple( blue& purple)
For ninhydrin test :
instantly it becomes purple and now dark purple
Before the bath it was fuzzy and now dark purple (changes color slowly)
Dark purple and like solid (changes color more slowly)
Grey& purple (not dissociate in water)
Change color to the purple slowly than glycine
For xanthoproeic test :
First fuzzy yellow then dark orange(orange&red)
Fuzzy white then fuzzy yellow
First light yellow, after orange ,then dark orange&Brown
For heavy metal precipitation test :
First, yellow. Then, light pink
Iron (III) nitrate
RESULTS AND DISCUSSIONS :
The aim was , determining whether amino acids or proteins are present in solution and amino acids are carrying a free amine group by using biuret test, ninhydrin test, xanthoproteic test and heavy metal precipitation test.
At first, the biuret test was showed that if there was 2 or more peptide or not.Cupper(II) ion was complexed with amine group (positive) gave the purple color. As a result of biuret test, it was identified that Casein amino acids contain double or more bond in their structure because they gave purple color and it shows that the test was positive.The ninhydrin test was showed if there was aldehyde,ammonia and free primary amine.The name of the complex was hydrindatin and the color was purple. The amino acids except casein has free amine group in their structure. The colors were generally dark purple
The xanthoproteic test showed if there was benzene or derivatives such as phenyl alanine, tyrosine , tryptophane,The color was yellow and tyrosine, tyrptophan, casein have yellow colour. So, it was understood that they have phenyl ring in their structure. The heavy metal test showed that if ions denaturates proteins similar with biuret and used iron(III) and AgNO3.
First sodium nitrate, silver nitrate and iron ( III) nitrate have same color yellow but the color of Silver nitrate was changed to light pink and the otherââ‚¬â„¢s color were same(yellow).